Transcript What is cell culture?

Animal Cell, Tissue and Organ Culture
What is cell culture?
 Cell culture refers to the removal of cells from a mammal or
an animal, and their subsequent growth in a favorable
artificial environment.
 The cells may be removed from the tissue directly and
disaggregated by enzymatic or mechanical means before
cultivation, or they may be derived from a cell line or cell
strain that has already been established.
 Basically, mammalian/animal cell culture techniques are
similar to those employed for bacteria, fungi, and yeast,
although there are some characteristic differences.
 In general, mammalian cells are more delicate, vulnerable
to mechanical damage, present lower growth rates, and
require more complex culture media and special substrates.
Cell Culture in vitro - A brief history
 1885: Roux maintained embryonic chick cells alive in saline
solution for short lengths of time.
 1912: Alexis Carrel cultured connective tissue and showed
heart muscle tissue contractility over 2-3 months.
 1943: Earle et al. produced continuous rat cell line.
 1962: Buonassisi et al. Published methods for maintaining
differentiated cells of tumour origin.
 1970: Gordon Sato et al. published the specific growth
factor and media requirements for many cell types.
 1979: Bottenstein and Sato defined a serum-free medium
for neural cells.
 1980: Chinese Hamster Ovary (CHO) cell lines were
developed. Recombinant erythropoietin was produced on
CHO cell by AMGEN (USA).
Acquiring cell cultures or cell lines
 Any laboratory may establish their own culture from primary
cells, or may choose to buy established cell cultures from
commercial or non-profit suppliers (i.e., cell banks).
 Various cell lines including those from insects, humans,
mice, rats, and other mammals are commercially available
to purchase.
 Reputable suppliers provide high quality cell lines that are
carefully tested for their integrity and to ensure that the
culture is free from any contaminants.
 Regardless of their source, it is advised to make sure that all
new cell lines are tested for mycoplasma contamination
before use them for any experiment.
Isolation of cell lines for in vitro culture
Tissue of interest
Cell or tissue culture in vitro
Primary culture
Sub-culture
Secondary culture
Sub-culture
Cell Line
Single cell isolation
Successive sub-culture
Immortalization
Clonal cell line
Loss of control
of cell growth
Transformed cell line
Immortalised cell line (cancerous cells)
Senescence
Finite vs Continuous Cell Line
 Normal cells usually divide only a limited number
of times before losing their ability to proliferate,
which is a genetically determined event known as
senescence; these cell lines are known as finite.
 However, some cell lines become immortal
through a process called transformation, which
can occur spontaneously or can be chemically or
virally induced. When a finite cell line undergoes
transformation and acquires the ability to divide
indefinitely, it becomes a continuous cell line.
Primary cell cultures or cell lines
 Primary culture refers to the stage of the culture after the
cells are isolated from the tissue and proliferated under
the appropriate conditions until they occupy all of the
available substrate (i.e., reach confluence) and retains
differentiated phenotype.
- At this stage, the cells have to be sub-cultured (i.e.,
passaged) by transferring them to a new vessel with
fresh growth medium to provide more room for
continued growth.
 Cell lines derived from primary cultures have a limited life
span (i.e., they are finite) and as they are passaged, cells
with the highest growth capacity predominate, resulting in
a degree of genotypic and phenotypic uniformity in the
population.
Secondary cultures
• Derived from a primary cell culture and
isolated by selection or cloning.
• Becoming a more homogeneous cell
population.
• Finite life span in vitro.
• Retain differentiated phenotype.
• Mainly anchorage dependant.
• Exhibit contact inhibition.
 Contact inhibition is a growth mechanism. In most
cases when two cells collide they attempt to move
in a different direction to avoid future collisions.
Contact inhibition of growth
Normal somatic cells
 Normal somatic cells when grown in culture will become
growth inhibited when they encounter another cell. The
cells in our bodies are governed by growth control
mechanisms and cellular senescence (aging). Cell aging
puts a limit on the number of times a cell can divide: the
more a cell has divided, the less likely it will be to divide
again. Growth mechanisms are in place to stop or
continue cell growth depending on the conditions.
Cancerous Cells
 Cancerous cells typically lose this property and thus grow
in an uncontrolled manner even when in contact with
neighboring cells. They aren't motivated to change
direction upon contact, so they pile up and grow over
each other.
Contact inhibition (contd.)
 In most cases, when two cells collide they attempt to move
in a different direction to avoid future collisions. As replication
increases the amount of cells, the number of directions those
cells can move without touching another is decreased.
 As the two cells come into contact, their locomotive process
is paralyzed. When the two cells colliding are different types
of cells, one or both may respond to the collision.
Continuous cultures
 Derived from a primary or secondary culture.
 Immortalised:
- Spontaneously (e.g.: spontaneous genetic mutation).
- By transformation vectors (e.g.: viruses &/or
plasmids).
 Serially propagated culture shows an increased growth
rate and shows homogeneous cell population.
 Loss of anchorage dependency and contact inhibition.
 Infinite life span in vitro.
 Differentiated phenotype: very little retained with
transformed cell lines (cancerous cells).
Cell lines used for in vitro culture are two types
1. Anchorage dependant
- Most animal derived cells.
- Adhere to bottom of a flask and form a monolayer.
- Eventually cover entire surface of substratum.
- Proliferation then stops.
- Need to subculture cells to fresh medium.
- Proliferation can begin again.
2. Anchorage independent
- Cells associated with body fluid (eg. blood cells).
- Grown in suspension.
- Will eventually need sub-culturing.
Mammalian cell morphology
Most mammalian cells in culture can be divided into three
basic categories based on their morphology:
1. Fibroblastic (or fibroblast-like) cells are bipolar or
multipolar, have elongated shapes and grow attached to the
surface of growth container.
2. Epithelial-like cells are polygonal in shape with more
regular dimensions and grow attached to the surface of
growth container in discrete patches.
3. Lymphoblast-like cells are spherical in shape and usually
grown in suspension without attaching to a surface.
Fibroblastic cells
Epithelial-like cells
Lymphoblast-like cells
Cell morphology (contd.)
• In addition to the basic categories mentioned in previous
slide, certain cells display morphological characteristics
specific to their specialized role in host.
• Moreover, neuronal cells exist in different shapes and
sizes, but they can roughly be divided into two basic
morphological categories, type I with long axons used to
move signals over long distances and type II without
axons. A typical neuron projects cellular extensions with
many branches from the cell body, which is referred to as
a dendritic tree.
Type I neuronal cells
Type II neuronal cells
Example of some cell lines used for in vitro culture
Cell
line
Species of
origin
Tissue of
origin
Cell
morphology
3T3
Mouse
Connective
Fibroblast
CHO
Chinese
Ovary
Epithelial
Kidney
Fibroblast
Cervical
Epithelial
Hamster
BHK21
Syrian
Hamster
HeLa
Human
Carcinoma
MDCK
Dog
Kidney
Epithelial
MRC-S
Human
Lung
Fibroblast,
Finite
Applications of Mammalian Cell Culture
 Cell culture is one of the major tools used in cellular and
molecular biology, providing excellent model systems for
studying the normal physiology and biochemistry of cells
(e.g., metabolic studies, aging), the effects of drugs and
toxic compounds on the cells, and mutagenesis as well as
carcinogenesis.
 It is also used in drug screening and development, and
large scale manufacturing of biological compounds (e.g.,
vaccines, therapeutic proteins).
 The major advantage of using cell culture for any of the
these applications is the consistency and reproducibility
of results that can be obtained from using a batch of
clonal cells.
What do the cells need to grow?
Culture conditions vary widely for each cell type, but the
artificial environment in which the cells are cultured
invariably consists of a suitable vessel containing a substrate
or medium that
- supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and
gases (O2, CO2), and
- regulates the physicochemical environment (pH, osmotic
pressure, temperature).
Anchorage dependent must be cultured while attached to a
solid or semi-solid substrate (adherent or monolayer
culture), while others can be grown floating in the culture
medium (suspension culture).
Sterility (aseptic technique, antibiotics and antimycotics)
- Mycoplasma contamination must be tested.
Humid CO2 incubator
 A good CO2 incubator is expensive.
 A controlled atmosphere is achieved by using a
humidifying tray and controlling the CO2 tension with
a CO2-monitoring device, which draws air from the
incubator into a sample chamber, determines the
concentration of CO2, and injects pure CO2 into the
incubator to make up any deficiency.
The incubator should be large
enough (ranging 50-200 liters,
and have forced air circulation,
temperature control and a safety
thermostat to protect from
overheating or poor heating.
Nutrients (culture media)
Basal Media are used to maintain pH and osmolarity (260320 mOsm/L) and provide nutrients and energy source. The
components of basal media are as follows.
Inorganic Salts
• Maintain osmolarity.
• Regulate membrane potential (Na+, K+, Ca2+).
• Provide ions for cell attachment and enzyme
cofactors.
pH Indicator - Phenol Red
• Optimum cell growth occurs at approx. pH 7.4
• Phenol red is used to monitor the changes from red
to yellow
Buffers (Bicarbonate and HEPES)
• Bicarbonate buffered media requires CO2
atmosphere. But, HEPES does not require CO2.
Nutrients (contd.)
Keto acids (oxalacetate and pyruvate)
- Intermediate in Glycolysis/Krebs cycle
- Keto acids added to the media as additional energy source
- Maintain maximum cell metabolism
Carbohydrates
- Energy source
- Glucose and galactose
- Low (1 g/L) and high (4.5 g/L) concentrations of sugars in
basal media
Vitamins
- Precursors for numerous co-factors
- B group vitamins necessary for cell growth and proliferation
- Common vitamins found in basal media are riboflavin,
thiamine and biotin
Trace Elements
- Zinc, copper, selenium and tricarboxylic acid intermediates
Supplements to basal media
L-glutamine
- Essential amino acid (not synthesised by the cell)
- Energy source (citric acid cycle), used in protein synthesis
- Unstable in liquid media - added as a supplement
Non-essential amino acids (NEAA)
- Energy source, used in protein synthesis
- May reduce metabolic burden on cells
Growth Factors and Hormones (e.g.: insulin)
- Stimulate glucose transport and utilization
- Uptake of amino acids
- Maintenance of differentiation
Antibiotics and Antimycotics
- Penicillin, streptomycin, gentamicin, amphotericin B
- Reduce the risk of bacterial and fungal contamination
- Cells can become antibiotic resistant - changing phenotype
- Preferably avoided in long term culture
Supplements to basal media (contd.)
Foetal Calf/Bovine Serum (FCS & FBS)
- Growth factors and hormones
- Aids cell attachment
- Binds and neutralise toxins
- Long history of use
Heat Inactivation (560C for 30 mins) - why?
- Destruction of immunoglobulins
- Destruction of some viruses (also gamma
irradiated serum)
 Care! Overdoing heat inactivation can damage growth
factors, hormones & vitamins and affect cell growth.
How do we culture cells in the laboratory?
Revive frozen cell population
Isolate from tissue
Containment level 2
cell culture laboratory
Maintain in culture
(aseptic technique)
Typical
cell culture flask
Sub-culture (passaging)
Count cells
‘Mr Frosty’
Used to freeze cells
Cryopreservation
Liquid nitrogen is
also used
Passaging Cells
Check confluency of cells
Remove spent medium
Wash with PBS
Incubate with
trypsin/EDTA
Why passage cells?

To maintain cells in culture (i.e. don’t overgrow)

To increase cell number for experiments/storage
How?

70-80% confluency

Wash in PBS to remove dead cells and serum

Trypsin digests protein-surface interaction to
release cells (collagenase is also useful)

EDTA enhances trypsin activity

Resuspend in serum (inactivates trypsin)

Transfer dilute cell suspension to new flask
(fresh media)

Most cell lines will adhere in approx. 3-4 hours
Resuspend in serum
containing media
Transfer to culture flask
70-80% confluence
100% confluence
Cryopreservation of cells
Passage cells
Resuspend cells in serum
containing media
Centrifuge &
Aspirate supernatant
Resuspend cells in
10% DMSO in FCS
Transfer to cryovial
Freeze at -800C
Transfer to liquid
nitrogen storage tank
Why cryopreserve cells?
• Reduced risk of microbial contamination.
• Reduced risk of cross contamination with
other cell lines.
• Reduced risk of genetic drift and
morphological changes.
• Research conducted using cells at
consistent low passage.
How?
• Log phase of growth and >90% viability
• Passage cells & pellet for media exchange
• Cryopreservant (DMSO) – precise
mechanism unknown but prevents ice
crystal formation
• Freeze at -80oC (‘slow’ freezing)
• Liquid nitrogen -1960C
Cell counting by hemacytometer
 Clean the chamber and cover
slip with alcohol. Dry and fix the
cover slip in position.
 Harvest the cells. Add 10 μL of
the cells to the hemacytometer.
Do not need to overfill.
 Place the chamber in the
inverted microscope under a 10X
objective. Use phase contrast to
distinguish the cells.
 Count the cells in the large,
central gridded square (1 mm2).
The gridded square is circled in
the graphic. Multiply by 104 to
estimate the number of cells per
mL. Prepare duplicate samples
and average the count.
Automated cell counter
Cellometer lets you:
• View cell morphology, for visual confirmation after cell
counting.
• Take advantage of 300+ cell types and easy, wizard-based
parameter set-up.
• Save sample images with results securely on your
computer, plus autosave results on the network for added
convenience and data protection.
Determining the cell viability
The following procedure will enable you to accurately determine the cell
viability. Cell viability is calculated as the number of viable cells divided by
the total number of cells within the grids on the hemacytometer. If cells take
up trypan blue, they are considered non-viable.
1. Determine the cell density of your cell line suspension using a
hemacytometer.
2. Prepare a 0.4% solution of trypan blue in buffered isotonic salt solution,
pH 7.2 to 7.3 (i.e., phosphate-buffered saline).
3. Add 0.1 mL of trypan blue stock solution to 1 mL of cells.
4. Load a hemacytometer and examine immediately under a microscope at
low magnification.
5. Count the number of blue staining cells and the number of total cells. Cell
viability should be at least 95% for healthy log-phase cultures.
% viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100
To calculate the number of viable cells per mL of culture, use the formula
below. Remember to correct for the dilution factor.
Number of viable cells × 104 × 1.1 = cells/mL culture
The ideal growth curve for cells in culture
Organ culture
 Not whole but pieces of organs can be cultured on artificial
medium. For organ culture, care should be taken to handle
in such a way that tissue should not be damaged.
 The culture media on which organ is cultured are the same
as described for cell and tissue culture. However, it is more
easy to culture embryonic organs than adult animals.
 Methods of culturing embryonic organ and adult organs
differ. Due to the requirement of high O2 amount (~95%),
special serum-free media (e.g. T8) and special apparatus
(Towell’s Type II culture chamber) are used for adult organ
culture.
 The embryonic organs can be cultured either on agar or in
liquid media.
Contamination
 A cell culture contaminant can be defined as some element in the
culture system that is undesirable because of its possible
adverse effects on either the system or its use.
Contamination must be avoided because
 They compete for nutrients with host cells and secrete acidic or
alkaline by-products that ceases the growth of the host cells.
 Degraded arginine & purine inhibits the synthesis of histone and
nucleic acid.
 They also produces H2O2 which is directly toxic to cells
Two types of contaminants
1. Chemicals - difficult to detect caused by impurities in media,
sera, water, endotoxins, plasticizers, metal ions and traces of
detergent that are invisible.
2. Biological contaminants
Biological contamination
2. Biological - contamination by microorganisms remains a major
problem in tissue culture. Bacteria, mycoplasma, yeast, and
fungal spores may be introduced via the operator, the
atmosphere, work surfaces, solutions, and many other sources.
A
B
C
Figure. Phase contrast microscopic images of adherent 293 cells
contaminated with E. coli (panel-B) and yeast (panel-C). Further
magnification of the area enclosed by the black square in panel-A
resolves the individual E. coli cells, which are typically rod-shaped
(about 2 μm long and 0.5 μm in diameter).
Biosafety Levels
According to the United States guidelines prepared by the Centers for
Disease Control (CDC), the National Institutes of Health (NIH), and the
Department of Health and Human Services (DHHS), there are four
ascending levels of containment, referred to as biosafety levels 1 through 4.
Biosafety Level 1 (BSL-1)
BSL-1 is the basic level of protection common to most research and clinical
laboratories, and is appropriate for agents that are not known to cause
disease in normal, healthy humans.
Biosafety Level 2 (BSL-2)
BSL-2 is appropriate for moderate-risk agents known to cause human
disease of varying severity by ingestion or through percutaneous or
mucous membrane exposure. Most cell culture labs should be at least BSL2, but the exact requirements depend upon the cell line used and the type
of work conducted.
Biosafety Level 3 (BSL-3)
BSL-3 is appropriate for indigenous or exotic agents with a known potential
for aerosol transmission, and for agents that may cause serious and
potentially lethal infections.
Biosafety Level 4 (BSL-4)
BSL-4 is appropriate for exotic agents that pose a high individual risk of lifethreatening disease by infectious aerosols and for which no treatment is
available. These agents are restricted to high containment laboratories.
How can cell culture contamination be controlled?
 Watch the video on
tissue culture and
safety practice.
Transgenic Animals
Methodology and Applications
Transgenic mice
 Hundreds of different genes have been introduced into
various mouse strains. These studies have contributed to an
understanding of
 gene regulation, tumor development, immunolo-gical
specificity, molecular genetics of development, and
many other biological processees of interest.
 Transgenic mice have also played a role in examining the
feasibility of the industrial production of human therapeutic
drugs by domesticated animals and in the creation of
transgenic strains that act as biomedical models for various
human genetic diseases.
Transgenic mice: methodology
 DNA can be introduced into mice by
1. retroviral vectors that infect the cells of an earlystage embryo prior to implantation into a respective
female,
2. microinjection into the enlarged sperm nucleus (male
pronucleus) of a fertilized egg, or
3. introduction of genetically engineered embryonic
stem cells into an early-stage developing embryo
before implantation into a respective female.
The retroviral vector method
 The use of retroviral vectors has the advantages of
being an effective means of integrating the transgene
into the genome of a recipient cell.
 Retroviruses have RNA genomes that are used as
templates for reverse transcriptase to synthesize a copy
of DNA that can be inserted into the host cell genome.
The retroviral vector method (contd.)
There are some drawbacks to the use of retroviral vectors.
 Vector derived from this viruses can transfer only small
pieces (~8 kb) of DNA.
 Although these vectors are designed to be replication
defective, the genome of the retroviral strain (helper virus)
that is needed to create large quantities of the vector DNA
can be integrated into the same nucleus as the transgene.
 It is absolutely necessary that there should not be any
retroviral contamination for applications in which either a
commercial product is to be synthesized by the transgenic
organism or the transgenic organism is to be used as food.
 In addition, transgene introduced on some retroviral vectors
are silenced in mouse embryos.
The lentiviral vector system
The lentivirus vector system is similar to other
retroviral vector systems and
 is capable of delivering large segments of
DNA into host genome,
 is stable for relatively long periods,
 has low immunogenicity, and
 can infect both dividing and non-dividing
cells.
Fig. Establishing transgenic
mice with retroviral vectors
 Cleavage stage embryos,
usually at the eight–cell stage,
are infected with a defective
retrovirus carrying a transgene.
 Implanted females (foster
mothers) give birth to transgenic
pups.
 Matings are carried out to
determine which pups have the
transgene in their germ line
cells.
 Transgenic lines can be
established from these founder
transgenic animals.
Fig. Establishing transgenic
mice by DNA microinjection
 Because of the disadvantages of the
retroviral vector method,
microinjection of DNA is currently the
preferred methods for producing
transgenic mice.
 Eggs are obtained from donor
females that have been induced to
superovulate and then mated with
males.
 Purified samples of the transgenic
construct are microinjected into the
male pronucleus of a fertilized egg.
 Implanted female gives birth to
transgenic pups, from which
transgenic lines can be established.
How to create superovulated female mouse?
 The number of available fertilized eggs that are to be
inoculated by microinjection is increased by stimulating
donor females to superovulate.
 Female mice are given an initial injection of pregnant
mare’s serum and another injection of human chorionic
gonadoprotein after about 48 hours.
 The superovulated female mice produces about 35 eggs
instead of the normal 5 to 10 that are subsequently
mated so that eggs become fertilized.
 Microinjection of the fertilized eggs has to be performed
immediately after their collection from oviducts.
Fig. Establishing transgenic mice
by DNA microinjection (contd.)
 In DNA microinjection method, the injected
DNA integrates at random sites within the
genome, and often multiple copies of the
injected DNA are incorporated in one site.
 Therefore, not all of the transgenic pups will
have the appropriate characteristic.
 Need to check mouse pups for DNA (by PCR
or Southerns), RNA (by northerns or RTPCR), and protein (by western or by some
specific assay method).
 In some individuals, the transgene may not be
expressed because of the site of integration,
and in others, the copy number may be
excessive and may lead to overexpression,
which disrupts the normal physiology of the
animal.
Fig. Overall efficiency of the transgenesis process
after DNA microinjection
 All the fertilized eggs
(100%) of cattle, pigs,
sheep and mice were
inoculated with a
transgene.
 The success of
implantation and
giving birth to
offspring is much
lower.
 Less than 5% or fewer
of the microinjected
fertilized eggs become
transgenic progeny
Establishing transgenic mice
with genetically engineered
embryonic stem (ES) cells
 An embryonic stem cell culture is
initiated from the inner cell mass of a
mouse blastocyst.
 The embryonic stem cells are
transfected with a transgene. After
growth, the transfected cells are
identified by either the positive-negative
selection procedure or PCR analysis.
 Populations of transfected cells are
cultured and inserted into blastocyst,
which are then implanted into foster
mothers.
 Transgenic lines can be established by
crosses from founder mice that carry
the transgene in their germ lines.
Positive and negative selection of recombinant ES cells
 When transfected cells are grown in the
presence of G-418, all the cells that
lack the NeoR gene are killed.
Therefore, only cells with integrated
DNA survive; i.e., these cells are
positively selected.
 If the compound ganciclovir is added at
the same time as G-418, the cells that
express thymidine kinase are killed
because thymidine kinase converts
ganciclovir to toxic compounds that kill
cells; i.e., these cells are negatively
selected.
 The cells most likely to survive this
dual-selection scheme are those that
have DNA integrated at the target site.
tkHSV = thymidine kinase from
herpes simplex virus
ES cells can be used to create knockout mutants
One of the aims of targeted
gene disruption (gene
knockout) is to determine
the developmental and
physiological consequences of inactivating a
particular gene.
In addition, a transgenic
line with a specific disabled
gene can be used as a
model system to study the
molecular pathology of
human disease.
Cloning sheep by nuclear
transfer.
 The nucleus of an ovum is
removed (dashed arrow) with
a pipette.
 Cells from the mammary
epithelium of an adult are
grown in culture.
 The G0 (quiescent, nondividing) state is induced by
inhibiting cell growth.
 A G0 cell and enucleated
ovum are fused, and the renucleated ovum implanted
into a foster mother, where
development proceeds.
“Hello Dolly”
 In the experiment
described by Wilmut et al.
(1997), 277 enucleated
ova were fused with G0
mammary cells, and 1 of
29 transferred early-stage
embryos produced a live
lamb.
And now there is pet cloning for a “small”
fee.
Nine-week-old "Little
Nicky" peers out from
her carrying case in
Texas. Little Nicky,
a cloned cat, was sold
to its new owner
by ”Genetic Savings
and Clone” for $50,000
in December 2004.
 August 07, 2008 Bernann McKinney
with one of the 5 puppies cloned from
Booger, her late pet pit bull. It cost
her $50,000. When Booger was
diagnosed with cancer, a grief-stricken
McKinney sought to have him cloned first by the now-defunct “Genetic
Savings and Clone”, and then by
South Korean company “RNL Bio”.
Transgenic livestock such
as cattle, sheep and goat
 Increase casein content in milk.
 Express lactase in milk (to
remove lactose) for lactose
intolerant individuals.
 Resistance to bacterial, viral, and
parasitic diseases
YFG = your favorite gene
Some human proteins expressed in the
mammary glands of transgenic animals
 Antithrombin III (the first transgenic animal drug, an
anticlotting protein, approved by the FDA in 2009).
 Erythropoietin (used to treat anaemia resulting from cancer
and chemotherapy).
 Human growth hormones such as hGH and somatotropin
(used to treat human growth deficiency in children and
chronic renal insufficiency)
 Monoclonal antibodies such as anti-lipopolysaccharide
(used to treat sepsis).
 Plasminogen activator such as urokinase type plasminogen
activator (used to treat acute myocardial infraction and acute
stroke).
Establishing transgenic
chickens by transfection of
isolated blastoderm cells
 Cells from blastoderm donors
are removed, tansfected with a
transgene, and inserted into
subgerminal space of an
irradiated recipient blastoderm.
 Some of the resulting chickens
may be chimeric.
 The chimeras that have the
transgene in germ line cells are
bred to established transgenic
lines.
Why transgenic
chickens?
 Resistance to viral,
bacterial, and coccidial
diseases.
 Better feed efficiency.
 Lower fat and cholesterol
levels in eggs.
 Better meat quality.
 Eggs with pharmaceutical
proteins in them.
Transgenic fish
 Genes are introduced into fertilized eggs by DNA
microinjection or electroporation.
 No need to implant the embryo; development is external.
 Genetically engineered for more rapid growth using the growth
hormone gene (salmon, catfish, tuna, etc.).
 Genetically engineered for greater disease resistance.
 Genetically engineered to serve as a biosensor for water
pollution.
 Genetically engineered for a novel pet (Glofish)
Transgenic fish (more detail)
 Salmon were genetically engineered for more rapid growth
using the growth hormone (GH) gene under the control of the
ocean pout antifreeze protein (AFP) gene promoter.
 Madaka fish were genetically engineered to serve as
biosensors for environmental pollutants (e.g., estrogens) by
using an estrogen-inducible promoter (the vitellogenin
promoter) to control expression of the GFP gene.